Risk Factors for Hypertension among Adult Patients Attending the General Outpatient Clinics of a Tertiary Hospital in Uyo, South-South Nigeria.

BACKGROUND: Hypertension is a modifiable risk factor for cardiovascular, cerebrovascular and renal diseases. OBJECTIVE: The objective of the study was to determine the risk factors for Hypertension among adults attending the general outpatient clinic of University of Uyo Teaching Hospital. METHODS: A cross-sectional study of three hundred and eightyfive (385) adults (18 years and above) attending the General Outpatient Clinic of the University of Uyo Teaching Hospital, Uyo, Nigeria, was carried out between March and June, 2013. Information on socio-demographic characteristics, presence or absence of hypertension, symptom counts, duration of illness as well as risk factors for hypertension were sought. RESULTS: The study 385 subjects consisted of 166 males and 219 females (male: female = 1:1.3). The mean age of respondents was 37.7± 14.4 years. After multivariate analysis, age, family history of hypertension and obesity were identified as independent risk factors for hypertension in this study. CONCLUSION: There is need for public enlightenment on the essence of routine screening of family members of hypertensive patients, avoidance of obesity and reduction of salt intake in our meals.

Detection and characterisation of parasites causing emerging zoonoses.

Understanding the epidemiology of zoonotic parasitic infections is dependent upon the availability of accurate and sensitive diagnostic techniques. The development of molecular diagnostic methods, particularly those utilising PCR for the detection of zoonoses will contribute greatly to the identification and control of these pathogens, by increasing the speed of diagnosis, specificity and sensitivity, reproducibility and ease of interpretation. Molecular characterisation studies allow us to distinguish between closely related infectious agents and to document the patterns of transmission of 'strains' and species within populations. This will allow precise determinations to be made about the aetiological agent, its characteristics and the source of infection. This review focuses on recent detection and characterisation techniques for both emerging and re-emerging parasite zoonoses.

Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture.

This study describes the complete development (from sporozoites to sporulated oocysts) of Cryptosporidium parvum (human and cattle genotypes) in the HCT-8 cell line. Furthermore, for the first time the complete life cycle was perpetuated in vitro for up to 25 days by subculturing. The long-term maintenance of the developmental cycle of the parasite in vitro appeared to be due to the initiation of the auto-reinfection cycle of C. parvum. This auto-reinfection is characterised by the production and excystation of new invasive sporozoites from thin-walled oocysts, with subsequent maintenance of the complete life cycle in vitro. In addition, thin-walled oocysts of the cattle genotype were infective for ARC/Swiss mice but similar oocysts of the human genotype were not. This culture system will provide a model for propagation of the complete life cycle of C. parvum in vitro.

Variation in Cryptosporidium: towards a taxonomic revision of the genus.

Cryptosporidium is an important cause of enteric disease in humans and other animals. Limitations associated with conventional diagnostic methods for cryptosporidiosis based on morphological features, coupled with the difficulty of characterising parasites isolated in the laboratory, have restricted our ability to clearly identify species. The application of sensitive molecular approaches has obviated the necessity for laboratory amplification. Such studies have found considerable evidence of genetic heterogeneity among isolates of Cryptosporidium from different species of vertebrate, and there is now mounting evidence suggesting that a series of host-adapted genotypes/strains/species of the parasite exist. In this article, studies on the molecular characterisation of Cryptosporidium during the last 5 years are reviewed and put into perspective with the past and present taxonomy of the genus. The predictive value of achieving a sound taxonomy for the genus Cryptosporidium with respect to understanding its epidemiology and transmission and controlling outbreaks of the disease is also discussed.

Molecular and phylogenetic characterisation of Cryptosporidium from birds.

Avian isolates of Cryptosporidium species from different geographic locations were sequenced at two loci, the 18S rRNA gene and the heat shock gene (HSP-70). Phylogenetic analysis of the sequence data provided support for the existence of a new avian species of Cryptosporidium infecting finches and a second species infecting a black duck. The identity of Cryptosporidium baileyi and Cryptosporidium meleagridis as valid species was confirmed. Also, C. baileyi was identified in a number of isolates from the brown quail extending the host range of this species.

Giardia duodenalis cysts of genotype A recovered from clams in the Chesapeake Bay subestuary, Rhode River.

Filter-feeding molluscan shellfish can concentrate zoonotic and anthroponotic waterborne pathogens. Cysts of Giardia sp. were detected by immunofluorescent antibodies in tissues of the clams Macoma balthica and M. mitchelli from Rhode River, a Chesapeake Bay (Maryland) subestuary. Molecular tests identified the cysts as Giardia duodenalis Genotype A, the most common genotype recovered from humans. Macoma clams are burrowers in mud or sandy-mud substrata and preferentially feed on the surface sediment layer. Waterborne Giardia cysts settle rapidly to the bottom in slow-moving waters and contaminate the sediment. Macoma clams do not have economic value, but can serve as biologic indicators of sediment contamination with Giardia sp. cysts of public health importance. These clams can be used for sanitary assessment of water quality.

Molecular characterization of Cryptosporidium isolates from humans and other animals using random amplified polymorphic DNA analysis.

Genetic variation in 25 Cryptosporidium isolates was analyzed using the random amplified polymorphic DNA (RAPD) technique. Simple reproducible polymorphisms were generated (using five primers) from Cryptosporidium DNA that was free of contaminating bacterial DNA. The results generated by four of the five primers were statistically correlated (P < 0.001). The combined data from three primers were used to construct a phenogram using Jaccard's distance. Four groupings could be distinguished. Two C. serpentis isolates from snakes formed a distinct group of their own, whereas C. parvum isolates were divided into two main groups: one containing most human isolates and the other containing mostly domestic animals plus two remaining human isolates. Due to the sensitivity of the RAPD technique, isolates can now be analyzed genetically, directly from fecal samples without further biological amplification. This represents a significant advance on current techniques.

RAPD (random amplified polymorphic DNA) analysis of Giardia DNA and correlation with isoenzyme data.

Fourteen Giardia duodenalis isolates were examined using the RAPD (random amplified polymorphic deoxyribonucleic acid) technique. Simple reproducible polymorphisms were generated using 3 different RAPD primers. The results generated by each primer were very similar and were significantly correlated with each other. These data were then compared to existing isoenzyme electrophoresis data on the same isolates. The RAPD data divided the isolates into 10 groupings or rapdemes while the isoenzyme data divided them into 10 similar zymodemes. Both methods grouped 4 isolates (BAH42, BAH44c9, BAH12c9 and BAH39c7), which comprised a phenotypically heterogeneous assemblage with respect to growth rate and metabolism, into similar groupings. The 2 methods were significantly correlated (P < 0.001). It will therefore be possible to use RAPD for the characterization of isolates of Giardia, and other parasites such as Cryptosporidium, which are refractory to cultivation in vitro.

Morphological and genetic characterisation of Cryptosporidium oocysts from domestic cats.

Faecal samples were collected from domestic cats in the metropolitan area of the city of Perth, Western Australia, and screened for the presence of Cryptosporidium by both microscopy and PCR. Of 162 samples screened, two were positive for Cryptosporidium (a prevalence of 1.2%). Sample Ct33 was from an 18-month-old female and sample Ct131 from a 12-month-old female. Morphological studies revealed oocysts with an average size of 4.6 x 4.0 microm, smaller in size than isolates typically seen in humans (5.0 x 4.5 microm). Sequence analysis of PCR products showed sequences from cat isolates to be different to previously sequenced human and calf isolates, with cat isolates exhibiting 8.1% sequence divergence from these isolates. Phylogenetic analysis grouped the cat isolates into a distinct group, separate from other C. parvum isolates and Cryptosporidium species. These results lend support to the existence of a cat-adapted Cryptosporidium strain or species.

Cryptosporidium canis n. sp. from domestic dogs.

Oocysts of Cryptosporidium, from the feces of a naturally infected dog and from an HIV-infected human, were identified as the previously reported canine genotype of Cryptosporidium parvum, hereafter referred to as Cryptosporidium canis n. sp. Also among the oocysts from the dog, a trace amount of C. parvum bovine genotype was detected. Cryptosporidium canis oocysts from both the dog and human were infectious for calves. Oocysts excreted by calf 1 (dog source) were approximately 90% C. canis and 10% C. parvum, whereas those excreted by calf 3 (human source) were 100% C. canis. Oocysts from calf 1 infected calf 2 resulting in excretion by calf 2 of oocysts approximately 90% C. parvum and 10% C. canis. Oocysts of C. canis were not infectious for BALB/c neonatal mice or immunosuppressed C57 juvenile mice, although all control mice became infected with the C. parvum Beltsville isolate. Oocysts of C. canis from calf 1 and the human were structurally indistinguishable from oocysts of the C. parvum Beltsville isolate (bovine). However, C. canis oocysts differed markedly at the molecular level from all known species of Cryptosporidium based on sequence data for the 18S rDNA and the HSP 70 gene. The differences in genetics and host specificity clearly differentiate C. canis as a new species.

Differentiation between human and animal isolates of Cryptosporidium parvum using rDNA sequencing and direct PCR analysis.

Sequence analysis of a polymerase chain reaction (PCR)-amplified 298-bp region of the Cryptosporidium parvum 18S rRNA gene was carried out on 10 human and 9 animal isolates. Eight of the 9 animal isolates and 3 human isolates displayed the recognition sequence TATATTT, whereas 7/10 human isolates exhibited the recognition sequence TTTTTTTTTTT. Sequence analysis of the ninth animal isolate, which was recovered from a Koala, revealed this isolate to be different from both human and animal isolates. The AT richness of the rDNA recognition sequences rendered them unsuitable for primer design and therefore a diagnostic randomly amplified polymorphic DNA fragment previously developed in our laboratory was also sequenced. Analysis of 2 human and 2 animal isolates again revealed distinct differences between animal and human isolates. On the basis of this sequence information, diagnostic primers were designed that could directly differentiate between animal and human isolates on the basis of the size of the PCR product. The ability to differentiate directly between human and animal isolates has important implications for studies of the transmission and zoonotic potential of this organism. These results also raise further doubts about the uniformity of the species C. parvum.

Phylogenetic relationships among isolates of Cryptosporidium: evidence for several new species.

Isolates of Cryptosporidium were characterized using nucleotide sequence analysis of the 18S rRNA and dihydrofolate reductase genes and also random-amplified polymorphic DNA analysis. Phylogenetic analysis confirmed the validity of the species of Cryptosporidium examined in this study such as Cryptospordium muris and Cryptosporidium baileyi, and also reinforced evidence from numerous researchers worldwide suggesting that Cryptosporidium parvum is not a single uniform species. The data obtained provided strong support for the validity of Cryptosporidium felis. Evidence suggests that the newly identified marsupial and pig genotypes may also be distinct and valid species, but biological studies are required for confirmation.

Detection of the Cryptosporidium parvum "human" genotype in a dugong (Dugong dugon).

The Cryptosporidium "human" genotype was identified in a paraffin-embedded tissue section from a dugong (Dugong dugon) by 2 independent laboratories. DNA sequencing and polymerase chain reaction/restriction fragment length polymorphism analysis of the 18S ribosomal RNA gene and the acetyl CoA synthethase gene clearly identified the genotype as that of the Cryptosporidium variant that infects humans. This is the first report of the human Cryptosporidium genotype in a nonprimate host.

Presence of double-stranded RNAs in human and calf isolates of Cryptosporidium parvum.

We examined the occurrence of 2 virus-like double-stranded (ds)RNAs in human and calf isolates of Cryptosporidium parvum senso latu and other microorganisms, including 7 other members of the genus. A total of 32 isolates of C. parium, 16 from humans (5 from acquired immune deficiency syndrome patients) and 16 from calves, were analyzed. Ethidium bromide staining, or Northern blot analysis, or reverse transcription/polymerase chain reaction, or all 3 methods, revealed that both genotype 1 and genotype 2 isolates of C. parvum possessed these dsRNAs. No other Cryptosporidium spp. or other organisms examined possessed these dsRNAs. Comparison analysis of partial cDNA sequences of dsRNAs from human and calf isolates revealed a high degree of similarity (>92% and >93% identical nucleotides for large and small dsRNAs, respectively). Slight, consistent differences in nucleotide sequences could be seen at select sites and were associated with an isolate being either genotype 1 or 2. Because of the widespread distribution of the dsRNAs, the similarity of these molecules between isolates, and high host specificity, these nucleic acids may prove to represent species-specific molecular markers for C. parvum. Evidence also suggests that the dsRNA can be utilized for molecular genotyping of C. parvum.

Morphological and molecular characterization of Giardia isolated from the straw-necked ibis (Threskiornis spinicollis) in Western Australia.

Following the first report of avian Giardia infection in Australia, isolates of the parasite recovered from naturally infected straw-necked ibis (Theskiornis spinicollis) were characterized using median body morphology, scanning electron microscopy, multilocus enzyme electrophoresis, random amplified polymorphic DNA (RAPD), and small subunit ribosomal RNA (SSU-rRNA) analyses. Results were compared with Giardia from other birds and mammals, and the extent of genetic diversity between a range of ibis isolates collected in Western Australia was determined. The ibis isolates of Giardia were genetically relatively homogeneous, which is in contrast to the extensive genetic heterogeneity often displayed by mammalian Giardia isolates. Morphologically, Giardia from ibis were similar to Giardia ardeae although they differed genetically and by the fact that the ibis isolates could not be established in in vitro culture. Sequence data of the DNA coding for the SSU-rRNA found a 96% homology between the ibis isolates from Western Australia and G. ardeae, suggesting that they represent distinct strains of the same species. In contrast, the ibis isolates were genetically and morphologically very different than Giardia duodenalis and Giardia muris from mammals.

Phylogenetic analysis of Cryptosporidium isolates from captive reptiles using 18S rDNA sequence data and random amplified polymorphic DNA analysis.

Sequence alignment of a polymerase chain reaction-amplified 713-base pair region of the Cryptosporidium 18S rDNA gene was carried out on 15 captive reptile isolates from different geographic locations and compared to both Cryptosporidium parvum and Cryptosporidium muris isolates. Random amplified polymorphic DNA (RAPD) analysis was also performed on a smaller number of these samples. The data generated by both techniques were significantly correlated (P < 0.002), providing additional evidence to support the clonal population structure hypothesis for Cryptosporidium. Phylogenetic analysis of both 18S sequence information and RAPD analysis grouped the majority of reptile isolates together into 1 main group attributed to Cryptosporidium serpentis, which was genetically distinct but closely related to C. muris. A second genotype exhibited by 1 reptile isolate (S6) appeared to be intermediate between C. serpentis and C. muris but grouped most closely with C. muris, as it exhibited 99.15% similarity with C. muris and only 97.13% similarity with C. serpentis. The third genotype identified in 2 reptile isolates was a previously characterized 'mouse' genotype that grouped closely with bovine and human C. parvum isolates.

Low prevalence of Cryptosporidium parvum in hospitalized children in Kota Bharu, Malaysia.

The aim of this prospective study was to determine the prevalence of Cryptosporidium parvum in hospitalized children in Kota Bharu, Malaysia. Over a 19 month study period, 258 stool samples were examined from 159 children; 109 with diarrhea and 50 controls without diarrhea. Modified Ziehl-Neelsen staining method and a polymerase chain reaction (PCR) assay were used to detect C. parvum and the samples were also examined for the presence of other intestinal parasites. Only 1 of the 109 (0.9%) children with acute diarrhea was positive for C. parvum by microscopy and PCR. Thirty-one percent of children were infested with other intestinal parasites, the most common being Ascaris lumbricoides and Trichuris trichiura. In conclusion, we found C. parvum to be an uncommon infective agent in hospitalized children with or without diarrhea in Kota Bharu, Malaysia.

Concurrent Cryptosporidium and parvovirus infections in a puppy.

Cryptosporidium parvum, an intestinal coccidian parasite, was isolated from faeces and intestinal biopsies of a 9-week-old puppy with acute parvoviral gastroenteritis. Gene sequence analysis identified a Cryptosporidium genotype not previously recorded in Australia. The puppy recovered after treatment with crystalloid fluids, synthetic and natural colloids and jejunostomy tube feeding.

Molecular and biological characterisation of Cryptosporidium in pigs.

OBJECTIVE: Genetic and biological characterisation of 12 isolates of Cryptosporidium from pigs and comparing them with Cryptosporidium isolates from humans and cattle. DESIGN: Cryptosporidium isolates from pigs were compared with those obtained from human and cattle using rDNA sequence analysis. The infectivity of two of the porcine isolates was determined in neonatal mice and the clinical history of the infected pigs recorded. RESULTS: Pig-derived isolates of Cryptosporidium exhibited two distinct genotypes; a porcine genotype and a bovine genotype, which is common to cattle and other livestock. The porcine genotype did not produce any infection in neonatal mice whereas the bovine genotype did. CONCLUSION: Two distinct genetically and biologically differing strains of Cryptosporidium appeared to be associated with acute diarrhoea in pigs. Whether Cryptosporidium was a primary or secondary pathogen is unclear but warrants further investigation. As the bovine genotype is known to infect humans, the results suggest that pigs can act as reservoirs of cryptosporidial infections for humans and other live-stock. The zoonotic potential of the pig-adapted genotype is uncertain and requires further study.

Cryptosporidium meleagridis in an Indian ring-necked parrot (Psittacula krameri).

OBJECTIVE: To perform a morphological and genetic characterisation of a Cryptosporidium infection in an Indian ring-necked parrot (Psittacula krameri) and to compare this with C meleagridis from a turkey. DESIGN: Tissue and intestinal sections from an Indian ring-necked parrot were examined microscopically for Cryptosporidium. The organism was also purified from the crop and intestine, the DNA extracted and a portion of the 18S rDNA gene amplified, sequenced and compared with sequence and biological information obtained for C meleagridis from a turkey as well as sequence information for other species of Cryptosporidium. RESULTS: Morphological examination of tissue sections from an Indian ring-necked parrot revealed large numbers of Cryptosporidium oocysts attached to the apical border of enterocytes lining the intestinal tract. Purified Cryptosporidium oocysts measured about 5.1 x 4.5 microns, which conformed morphologically to C meleagridis. The sequence obtained from this isolate was identical to sequence information obtained from a C meleagridis isolate from a turkey. CONCLUSION: Cryptosporidium meleagridis was detected in an Indian ring-necked parrot using morphological and molecular methods. This is the first time that this species of Cryptosporidium has been reported in a non-galliform host and extends the known host range of C meleagridis.